Neuroscience_technics
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Ultrafast tissue staining with chemical tags

Ultrafast tissue staining with chemical tags | Neuroscience_technics | Scoop.it

Cellular and subcellular structures in thick biological samples typically are visualized either by genetically encoded fluorescent proteins or by antibody staining against proteins of interest. However, both approaches have drawbacks. Fluorescent proteins do not survive treatments for tissue preservation well, are available in only a few colors, and often emit weak signals. Antibody stainings are slow, do not penetrate thick samples well, and often result in considerable background staining. We have overcome these limitations by using genetically encoded chemical tags that result in rapid, even staining of thick biological samples with high-signal and low-background labeling. We introduce tools for flies and mice that drastically improve the speed and specificity for labeling genetically marked cells in biological tissues.(...) - by Kohl J et al., PNAS, vol. 111 no. 36, E3805–E3814, doi: 10.1073/pnas.1411087111

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A new nonscanning confocal microscopy module for functional voltage-sensitive dye and Ca2+ imaging of neuronal circuit activity

Recent advances in fluorescent confocal microscopy and voltage-sensitive and Ca2+ dyes have vastly improved our ability to image neuronal circuits. However, existing confocal systems are not fast enough or too noisy for many live-cell functional imaging studies. Here, we describe and demonstrate the function of a novel, nonscanning confocal microscopy module. The optics, which are designed to fit the standard camera port of the Olympus BX51WI epifluorescent microscope, achieve a high signal-to-noise ratio (SNR) at high temporal resolution, making this configuration ideal for functional imaging of neuronal activities such as the voltage-sensitive dye (VSD) imaging. The optics employ fixed 100- × 100-pinhole arrays at the back focal plane (optical conjugation plane), above the tube lens of a usual upright microscope. The excitation light travels through these pinholes, and the fluorescence signal, emitted from subject, passes through corresponding pinholes before exciting the photodiodes of the imager: a 100- × 100-pixel metal-oxide semiconductor (MOS)-type pixel imager with each pixel corresponding to a single 100- × 100-μm photodiode. This design eliminated the need for a scanning device; therefore, acquisition rate of the imager (maximum rate of 10 kHz) is the only factor limiting acquisition speed. We tested the application of the system for VSD and Ca2+ imaging of evoked neuronal responses on electrical stimuli in rat hippocampal slices. The results indicate that, at least for these applications, the new microscope maintains a high SNR at image acquisition rates of ≤0.3 ms per frame. (...) - by Tominaga T & Tominaga Y, Journal Of Neurophysiologie July 15, 2013 vol. 110 no. 2 553-561

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Neurient: An algorithm for automatic tracing of confluent neuronal images to determine alignment

Neurient: An algorithm for automatic tracing of confluent neuronal images to determine alignment | Neuroscience_technics | Scoop.it
  • We present Neurient, an algorithm developed to trace dense fluorescent neurite images.
  • Neurient allows unsupervised image processing, with optional tunable parameters.
  • Method: find seed points, index into a directed lookup table, trace neurite segments.
  • Trace coordinates and orientations can be used to evaluate neurite alignment. 
  • The Neurient code is open source and freely available for use and customization.

by Mitchel JA et al., Journal of Neuroscience MethodsVolume 214, Issue 2, 15 April 2013, Pages 210–222

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Encoded multisite two-photon microscopy

Encoded multisite two-photon microscopy | Neuroscience_technics | Scoop.it

The advent of scanning two-photon microscopy (2PM) has created a fertile new avenue for noninvasive investigation of brain activity in depth. One principal weakness of this method, however, lies with the limit of scanning speed, which makes optical interrogation of action potential-like activity in a neuronal network problematic. Encoded multisite two-photon microscopy (eMS2PM), a scanless method that allows simultaneous imaging of multiple targets in depth with high temporal resolution, addresses this drawback. eMS2PM uses a liquid crystal spatial light modulator to split a high-power femto-laser beam into multiple subbeams. To distinguish them, a digital micromirror device encodes each subbeam with a specific binary amplitude modulation sequence. Fluorescence signals from all independently targeted sites are then collected simultaneously onto a single photodetector and site-specifically decoded. We demonstrate that eMS2PM can be used to image spike-like voltage transients in cultured cells and fluorescence transients (calcium signals in neurons and red blood cells in capillaries from the cortex) in depth in vivo. These results establish eMS2PM as a unique method for simultaneous acquisition of neuronal network activity. (...) - by Ducros M et al., PNAS August 6, 2013 vol. 110 no. 32 13138-13143

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Seeing the forest tree by tree: super-resolution light microscopy meets the neurosciences

Seeing the forest tree by tree: super-resolution light microscopy meets the neurosciences | Neuroscience_technics | Scoop.it

Light microscopy can be applied in vivo and can sample large tissue volumes, features crucial for the study of single neurons and neural circuits. However, light microscopy per se is diffraction-limited in resolution, and the substructure of core signaling compartments of neuronal circuits—axons, presynaptic active zones, postsynaptic densities and dendritic spines—can be only insufficiently characterized by standard light microscopy. Recently, several forms of super-resolution light microscopy breaking the diffraction-imposed resolution limit have started to allow highly resolved, dynamic imaging in the cell-biologically highly relevant 10–100 nanometer range ('mesoscale'). New, sometimes surprising answers concerning how protein mobility and protein architectures shape neuronal communication have already emerged. Here we start by briefly introducing super-resolution microscopy techniques, before we describe their use in the analysis of neuronal compartments. We conclude with long-term prospects for super-resolution light microscopy in the molecular and cellular neurosciences.(...) - by Marta Maglione & Stephan J SigristNature Neuroscience 16, 790–797, 25 June 2013

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Two-Photon Excitation STED Microscopy in Two Colors in Acute Brain Slices

Two-Photon Excitation STED Microscopy in Two Colors in Acute Brain Slices | Neuroscience_technics | Scoop.it

Many cellular structures and organelles are too small to be properly resolved by conventional light microscopy. This is particularly true for dendritic spines and glial processes, which are very small, dynamic, and embedded in dense tissue, making it difficult to image them under realistic experimental conditions. Two-photon microscopy is currently the method of choice for imaging in thick living tissue preparations, both in acute brain slices and in vivo. However, the spatial resolution of a two-photon microscope, which is limited to ∼350 nm by the diffraction of light, is not sufficient for resolving many important details of neural morphology, such as the width of spine necks or thin glial processes. Recently developed superresolution approaches, such as stimulated emission depletion microscopy, have set new standards of optical resolution in imaging living tissue. However, the important goal of superresolution imaging with significant subdiffraction resolution has not yet been accomplished in acute brain slices. To overcome this limitation, we have developed a new microscope based on two-photon excitation and pulsed stimulated emission depletion microscopy, which provides unprecedented spatial resolution and excellent experimental access in acute brain slices using a long-working distance objective. The new microscope improves on the spatial resolution of a regular two-photon microscope by a factor of four to six, and it is compatible with time-lapse and simultaneous two-color superresolution imaging in living cells. We demonstrate the potential of this nanoscopy approach for brain slice physiology by imaging the morphology of dendritic spines and microglial cells well below the surface of acute brain slices. (...) - by Bethge P. et al., Biophysical JournalVolume 104, Issue 4, 778-785, 19 February 2013

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